In vivo trapping of FtsH substrates by label‐free quantitative proteomics

Abstract

FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli

“…Moreover, the novel Lon substrate MetR (see Figure C) also was among the trapped proteins. As previously observed in FtsH trapping experiments, we also trapped substrates of other proteases . FtsZ and IscS are substrates of ClpXP and FtsH, respectively …”

“…The global impact of Lon was analyzed by comparative proteomics between a lon‐ deficient and complemented strain using a quantitative Super‐SILAC approach . For identification of novel substrates we exploited a trapping approach previously shown to be successful for various proteases …”

“…For example, degradation of SoxS is known to be slowed down by an N‐terminal tag . It is important to note, however, that the ASKA collection has already proven to be suitable for the identification of FtsH substrates and revealed several new Lon substrates (Figure ) …”

“…Trapping experiments are based on proteolytically inactive protease variants with an active unfoldase, which captures and translocates substrates in vivo. Such experiments have previously been done with E. coli ClpXP, FtsH, and Lon . However, only a subset of the known E. coli Lon targets, namely RecA, RuvB, and IbpA were reported .…”

Structural Insights into <em>Thermotoga maritima</em> FtsH Periplasmic Domain on Substrate Recognition

“…In Escherichia coli, FtsH substrates in the inner membrane have been identified [11][12][13]. E. coli FtsH is responsible for the rapid degradation of the unassembled components of SecY inside the membrane [2,14], ensuring that the SecY translocase is always stoichiometrically assembled with the correct subunits for co-translational insertion and folding of membrane proteins.…”